We have continued our studies of chromatin structure, in order to determine how DNA is packaged within eukaryotic nuclei and, in particular, to learn how that structure is altered in the neighborhood of genes that are being expressed. We have investigated the solubility properties of chromatin from expressed and inactive genes, and shown that certain solubility differences thought by others to be intrinsic to transcriptionally active chromatin are in fact an indirect reflection of differences in sensitivity to nucleases. We have also continued our studies of the structure of chromatin in the neighborhood of the chicken adult Beta globin gene, isolated from the nuclei of erythrocytes in which the gene is being expressed. We had shown earlier that the chromatin containing the promoter region of this gene is unusually sensitive to nucleases, and we identified protein factors present in erythrocyte nuclei capable of conferring nuclease hypersensitivity on reconstituted complexes with Beta globin DNA. We have now determined precisely the binding sites and affinities of these factors. We find that binding is very tight and highly specific. Using "footprinting" methods in whch the pattern of protection against nuclease digestion is used to determine binding sites of proteins to DNA in vitro, we have shown that there are two distinct domains of binding, each about 25 base pairs long, within the 5' flanking region of the gene. Each domain is capable of binding protein independently of the other. We have shown that the factors are absent from similarly fractionated material isolated from oviduct, a tissue in which the globin gene is not expressed. In order to compare our in vitro studies with chromatin structure in vivo, we have developed a novel method of footprinting in vivo which allows us to determine directly the pattern of protein binding that occurs within the nucleus. The pattern we observe is quite similar to that we find with the complexes formed in vitro with partially purified erythrocyte nuclear factors, showing that these factors are bound specifically to the globin promoter region within the living erythrocyte nucleus.